Astrocytes play a fundamental role in ALS pathogenesis, participating in motor neuron (MN) degeneration in a non-cell autonomous manner. In spinal astrocytes from the G93A SOD1 mouse model of familial ALS, we found that ER oxidative stress induces post-translational changes in regulatory elements of ER calcium homeostasis (store operated calcium entry, SOCE), which leads to aberrant regulation of ER calcium levels and signaling. The resulting ER calcium signaling dysregulation participates in the toxic processes. In preparation to this application, we have studied human iPSC-derived SOD1 mutant astrocytes and found that aberrant ER calcium regulation is a common abnormal phenotype between the human cells and the mouse model of familial ALS, supporting its significance in ALS pathogenesis. Based on new preliminary studies, we hypothesize that ER calcium dysregulation in ALS astrocytes originates from enhanced oxidative protein folding process operated by the PDI-Ero1 enzyme pathway. Increased oxidative protein folding activity leads to excessive hydrogen peroxide production, glutathione consumption, and overburdening of molecular chaperones. We propose that this aberrant process causes oxidative modifications of proteins involved in calcium homeostasis. We postulate that these molecular mechanisms in ALS astrocytes are at the basis of the functional changes in intracellular calcium regulation and contribute to MN toxicity. The overarching goal of this application is to provide proof of principle that modulating ER calcium dysregulation pathways in ALS astrocytes represents a viable approach for improving ALS. To achieve this goal we propose three specific aims, which investigate different levels of regulation of ER calcium signaling and test the impact of modulating these pathways by pharmacological and genetic approaches on the toxicity of ALS astrocytes to MNs.